S Tantawy and others SF1 in Egyptians with 46,XY DSD 170:5 759–767 Clinical Study

Analysis of the coding for steroidogenic factor 1 (SF1, NR5A1) in a cohort of 50 Egyptian patients with 46,XY disorders of sex development

Sally Tantawy1,2, Inas Mazen2, Hala Soliman3, Ghada Anwar4, Abeer Atef4, Mona El-Gammal2, Ahmed El-Kotoury2, Mona Mekkawy5, Ahmad Torky2, Agnes Rudolf1, Pamela Schrumpf1, Annette Gru¨ ters1, Heiko Krude1, Marie-Charlotte Dumargne6, Rebekka Astudillo1, Anu Bashamboo6, Heike Biebermann1 and Birgit Ko¨ hler1

1Institute of Experimental Paediatric Endocrinology, University Children’s Hospital, Charite´ , Humboldt University, Correspondence Berlin, Germany, 2Department of Clinical Genetics and 3Department of Medical Molecular Genetics, Division of should be addressed Human Genetics and Genome Research, National Research Centre, Cairo, Egypt, 4Department of Paediatrics, to S Tantawy Cairo University, Cairo, Egypt, 5Department of Cytogenetics, Division of Human Genetics and Genome Research, Email National Research Centre, Cairo, Egypt and 6Human Developmental Genetics, Institut Pasteur, Paris, France [email protected]

Abstract

Objective: Steroidogenic factor 1 (SF1, NR5A1) is a key transcriptional regulator of involved in the hypothalamic– pituitary–gonadal axis. Recently, SF1 mutations were found to be a frequent cause of 46,XY disorders of sex development (DSD) in humans. We investigate the frequency of NR5A1 mutations in an Egyptian cohort of XY DSD. Design: Clinical assessment, endocrine evaluation and genetic analysis of 50 Egyptian XY DSD patients (without adrenal insufficiency) with a wide phenotypic spectrum. Methods: Molecular analysis of NR5A1 gene by direct sequencing followed by in vitro functional analysis of the European Journal of Endocrinology two novel missense mutations detected. Results: Three novel heterozygous mutations of the coding region in patients with hypospadias were detected. p.Glu121AlafsX25 results in severely truncated , p.Arg62Cys lies in DNA-binding zinc finger, whereas p.Ala154Thr lies in the hinge region of SF1 protein. Transactivation assays using reporter constructs carrying promoters of anti-Mu¨ llerian hormone (AMH), CYP11A1 and TESCO core enhancer of Sox9 showed that p.Ala154Thr and p.Arg62Cys mutations result in aberrant biological activity of NR5A1. A total of 17 patients (34%) harboured the p.Gly146Ala polymorphism. Conclusion: We identified two novel NR5A1 mutations showing impaired function in 23 Egyptian XY DSD patients with hypospadias (8.5%). This is the first study searching for NR5A1 mutations in oriental patients from the Middle East and Arab region with XY DSD and no adrenal insufficiency, revealing a frequency similar to that in European patients (6.5–15%). We recommend screening of NR5A1 in patients with hypospadias and . Yearly follow-ups of gonadal function and early cryoconservation of sperms should be performed in XY DSD patients with NR5A1 mutations given the risk of future fertility problems due to early gonadal failure.

European Journal of Endocrinology (2014) 170, 759–767

www.eje-online.org Ñ 2014 European Society of Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/EJE-13-0965 Printed in Great Britain

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Introduction Subjects and methods

Disorders of sex development (DSDs) are complex Cohort disorders with atypical chromosomal, gonadal or ana- A cohort of 50 Egyptian 46,XY DSD patients were recruited tomical sex (1). DSDs are classified into three groups: from the Pediatric Endocrinology Clinic of Cairo i) DSD with numerical sex chromosomal aberrations, University and the Clinical Genetics and Endocrinology ii) XY DSD and iii) XX DSD. Sex DSD Departments of National Research Centre (NRC) in Cairo, consists mainly of disorders with gonadal dysgenesis due Egypt. Patients’ chronological ages ranged from 2 months to sex chromosome imbalances such as Turner syndrome to 33 years at first presentation. The phenotypic spectrum (45,X and mosaicism), Klinefelter syndrome (47,XXY), included complete female external genitalia with/without mixed gonadal dysgenesis (45,X0/46,XY) and chimeric uterus (nZ8), ambiguous genitalia without uterus (nZ5), DSD (46,XX/46,XY). XY DSD comprises mainly testicular vanishing/atrophic testes (nZ10), hypospadias with nor- dysgenesis, defects of androgen synthesis or action and mally descended testis (nZ9), hypospadias with at least hypospadias. XX DSD includes congenital adrenal one undescended testis (nZ14) and isolated hypoplastic hyperplasia, ovarian dysgenesis and uterine/vaginal nZ malformations (1). In Egypt, the occurrence of DSD is phallus ( 4). Patients with syndromic forms of gonadal rare with an incidence of 1:5000 (2) similar to 1:4500 dysgenesis or chromosomal abnormalities were excluded. worldwide (3). In Egypt, 46,XY DSD constitutes the Among the 50 patients, 39 were reared as males, 11 as majority of DSD cases (65.9%) (4) with androgen females and one was reared as female at birth then her sex insensitivity syndrome and 5-a-reductase deficiency was changed to male at 2 years of age. Written informed being the two most frequent 46,XY DSD aetiologies due consent for genetic analysis was obtained according to to high consanguinity. They constitute 64% among all institutional ethical guidelines from the patients and/or reported cases (5). However, in many cases the patho- their parents. genetic cause is unknown. Work-up included cytogenetic analysis by G-banding During the last 10 years, steroidogenic factor 1 (SF1, technique in 50 metaphases (20), and pelviabdominal NR5A1) has been found to play a pivotal role in human sex ultrasound. , precursors and dihydrotesto- differentiation. SF1 is a key transcriptional regulator of sterone (DHT) were measured before and after human many genes involved in the hypothalamic–pituitary– chorionic gonadotropin (hCG) stimulation. In some cases, gonadal axis and (6). Sf1 null mice pelvic laparoscopy, biopsy and histological analysis of the

European Journal of Endocrinology demonstrate complete gonadal dysgenesis and adrenal were performed. failure (7). However, humans harbouring heterozygous NR5A1 mutations display a milder . The Molecular analysis of NR5A1 gene majority of cases display 46,XY DSD with ambiguous genitalia or hypospadias due to partial gonadal dysgenesis Molecular analysis of exons 2–7 of the gene encoding SF1 but no adrenal insufficiency. To date, about 60 different (NR5A1) was performed as described previously (21). NR5A1 mutations have been reported in humans with Numbering of the mutations is based on GenBank reference DSDs. So far, there is no apparent genotype–phenotype DNA sequence NM_004959.4, with the A of the ATG correlation in patients with NR5A1 mutations. initiation codon designated C1(www.hgvs.org/mutnomen). The phenotypic spectrum has been extended, involving A total of 100 Egyptian controls (200 alleles) were not only ambiguous genitalia and hypospadias due to sequenced for both the mutations and the single- gonadal dysgenesis (8, 9, 10), but also vanishing testis nucleotide polymorphisms (SNPs). syndrome (11), isolated hypoplastic penis (12) and male infertility (13, 14). Moreover, NR5A1 mutations were also Functional analysis found in 46,XX females with premature ovarian failure and primary ovarian insufficiency (15, 16, 17, 18, 19). Vector containing full-length mouse Nr5a1,pCNA3- Altogether, NR5A1 mutations have emerged as being the NR5A1- and the mouse TESCO reporter were a gift most frequent cause (6.5–15%) of different of from Dr Francis Poulat, Institut de Ge´ne´tique Mole´culaire 46,XY DSD in Western countries. Our aim is to investigate de Montpellier. The reporters containing the minimal whether NR5A1 mutations are also a cause of 46,XY DSD anti-Mu¨llerian hormone (AMH) and CYP11A1 promoters in Egypt. (K269) are described previously (17).

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A R62CR62C hypospadias: heterozygous p.Arg62Cys (c.184 COT), HumanHuman E S Q S C K I D K T Q R K R C P F C R F Q K C L heterozygous p.Glu121AlafsX25 (c.361delGAGACAGG) MutatedMutated Q C K R C P F C R F Q K C and heterozygous p.Ala154Thr (c.460 GOA) (Figs 1 ChimpChimp E S Q S C K I D K T Q R K R C P F C R F Q K C and 2). No NR5A1 mutations were found in patients RhesusRhesus Q R K R C P F C R F Q K C with other phenotypes such as complete gonadal dysgen- MouseMouse Q R K R C P F C R F Q K C esis, isolated micropenis or vanishing/atrophic testes. XenopusXenopus Q R K R C P Y C R F Q K C ElegansElegans A E A N C H V D R T C R K R C P S C R F The consanguinity rate among parents of all included DrosophilaDrosophila A E R S C H I D K T Q R K R C P Y C R 46,XY DSD patients in our study was 53%, where 34% were first cousins and 19% were second cousins or further relatives. In Egypt, the rate of total consanguineous A154TA154T B marriages was 29.7% in 2008 (22) and reached 35.3% in HumanHuman S L H G P E P K G L A A G P P A G P L G D F G A 2011 (23). MutatedMutated S L H G P E P K G L A T G P P A G P L G D F G ChimpChimp S L H A P E P K G L A A G P P A G P L G D F G RhesusRhesus S L H A P E P K G L A A G P P T G P L G D F G Case histories MouseMouse S L H A P E P K A L V S G P P S G P L G D F G XenopusXenopus N I H T I H PVTPVT K N L P P N PAPAP M T P VEYDRVEYDR G P Y G Patient 1 (p.Arg62Cys) " Patient 1 is a 1 six and a half- DrosophilaDrosophila E I Q I P Q V S S L T Q S P D S S P year-old male, born to non-consanguineous parents, presenting with severely hypoplastic phallus (stretched Figure 1 penile length was 1 cm, !K2.5 SDS), penile hypospadias, (A) The mutated arginine residue in position 62 is highly hypoplastic scrotum and impalpable gonads. Pelviabdom- conserved in homologues of SF1 from all species studied to inal ultrasound revealed two small testes, each 8–9 mm in date. (B) The mutated alanine position 154 is conserved in diameter, in medial ends of respective inguinal canals and human, chimp and rhesus but not in other species. no Mu¨llerian structures. The patient was operated upon for orchidopexy, correction of hypospadias and circumci- The NR5A1 expression vectors containing the sion. During mini puberty, basal testosterone level was low p.Ala154Thr, p.Arg62Cys and p.Gly35Glu variants were normal for age, while testosterone peak after hCG generated by site-directed mutagenesis (QuikChange, stimulation (5000 IU/m2 divided over 3 days) was mark- Stratagene) of pCNA3-NR5A1-myc plasmid. The entire edly low. Luteinising hormone-releasing hormone (LHRH) coding sequence of all mutant plasmids was confirmed by stimulation test showed slightly elevated FSH after European Journal of Endocrinology direct sequencing prior to functional studies. Transient gene stimulation. AMH and inhibin B were both diminished expression assays to assess NR5A1 function were performed (Table 1). The patient showed no clinical signs or in 96-well plates (TPP) using human embryonic kidney cells symptoms of adrenal affection and had normal cortisol (HEK293-T), FuGENE6 and a Dual-Luciferase Reporter Assay and DHEAS levels. Mutational analysis revealed a hetero- System (Promega) with pCMV-RL Renilla luciferase (Pro- zygous p.Arg62Cys (c.184 COT) mutation which lies in mega) expression as a marker of transfection efficiency. theDNA-bindingzincfingerregioninexon3and pCNA3-NR5A1-myc WT or mutant expression vector were co-transfected into HEK293-T cells with reporter and Zinc A pCMV-RL. Cells were lysed 48 h later and luciferase assays fingers box Hinge Ligand-binding domain AF-2 were performed (Dual-Luciferase Reporter Assay system, 1 461 Promega) using Centrox3 LB960 (Berthold Technologies). DNA-binding domain All data were standardised for Renilla activity. Results are R62C A154T G E121Afs*25 R313C shown as the mean S.E.M.oftwotothreeindependent V20L Y183X W279X experiments, each performed in 12 replicates. The data were E11X Q107X analysed statistically using Student’s t-test. A P value !0.05 was considered statistically significant. Figure 2 Cartoon structure of SF1 protein showing the location of three mutations in our study (boxed) and previously reported NR5A1 Results mutations in patients with hypospadias (grey) (E11X and NR5A1 analysis revealed three novel heterozygous Q107X, Kohler et al. (8); Y183X and W279X, Warman et al. (9); mutations of the coding region in three patients with R313C, Allali et al. (10); V20L and Camats et al. (16)).

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Table 1 Clinical, hormonal and molecular data of patients with NR5A1 mutations.

Epididymis, Adrenal Age External vas Gender affection/ Mutations Patient (years) genitalia Gonads Uterus deferens Hormones assignment investigations (heterozygous) Parents

1 1 6/12 Penile hypo- Bilateral small No Yes At 4 months: M Cortisol, 9.9 mg/dl p.Arg62Cys NA spadias, inguinal testes testosterone, (N) (exon 3)C severely hypo- (each 8–9 mm in 0.1–1.8 ng/ml (Y); DHEAS, 12.4 mg/dl p.Gly146Ala plastic penis diameter) FSH, 1.3–9.1 U/l (N) (exon 4) DSD 46,XY with Egyptians in SF1 others and Tantawy S (1 cm) and ([) and LH, hypoplastic 0.5–2.2 U/l (N) scrotum At 3 years: inhibin B, 15.5 ng/l (Y) and AMH, 10.6 ng/ml (Y) 2 13 Hypospadias, Right atrophic No Yes At 13 years: testo- M No symptoms p.Glu121AlafsX25 Mother severely hypo- undescended sterone post- of the adrenals (exon 4) WT plastic penis testis hCG, 0.75 ng/ml being affected Father WT (3.5 cm) and Left testis normal (Y); inhibin B, bifid scrotum in scrotum 9.0 ng/l (Y) and AMH, ! 0.10 ng/ml (Y) 3 4 Penile hypo- Both testes No Yes At 4 years: testo- M No symptoms p.Ala154Thr Mother spadias, hypo- normal in sterone, of the adrenals (exon 4) WT plastic penis scrotum 0.18–1.6 ng/ml (Y); being affected Father (2.5 cm) and FSH, 1.2–8.7 p.Al- anchored U/l (N); LH, a154Thr 0.6–2.1 U/l (N); inhibin B, 70.4 ng/l (N) and AMH, O20 ng/ml (N) Downloaded fromBioscientifica.com at09/27/202104:16:59AM

Conversion to SI units: testosterone (ng/mL)!3.47Z(nmol/l); FSH and LH (mIU/ml)!1Z(IU/l); AMH (ng/ml)!7.14Z(pmol/l) and DHEAS (mg/dl)!0.027Z(mmol/l). Serum testosterone levels are basal and after hCG stimulation. FSH and LH levels are basal and 90 min after LHRH stimulation. Normal values: basal testosterone level in males with: Tanner stage 1 (!7 years), !0.11–0.65; Tanner stages 2–3, 0.62–2.26 ng/ml and Tanner stages 4–5, 1.68–6.0 ng/ml. Normal testosterone peak after stimulation with 5000 IU/m2 hCG at 72 h: 3.0–10.0 ng/ml. Basal FSH in males: Tanner stage 1 (0.5–10 years), ! O ! O !

1–1.3 IU/l; Tanner stages 2–3 ( 10 years), 1–4.0 IU/l and Tanner stages 4–5 ( 10 years), 1.4–5.1 IU/l. After LHRH stimulation, FSH in males: Tanner stage 1, 1.1–6.3 IU/l; Tanner stages 2–3, 170 !4 IU/l and Tanner stages 4–5, 4–15.2 IU/l. Basal LH in males: Tanner stage 1, !1–1.5 IU/l; Tanner stages 2–3, 1–4.1 IU/l and Tanner stages 4–5, 3.4–7.5 IU/l. After LHRH stimulation, LH in males:

Tanner stage 1, !1.4–4.1 IU/l; Tanner stages 2–3, 1.5–4.1 IU/l and Tanner stages 4–5, 19–37 IU/l. Inhibin B reference values: see reference (25). AMH (26): in childhood, medianZ105.7 ng/ml and :5 rangeZ55.3–186.9 ng/ml; prepubertal !9 yearsZ97.58 (44.94–170.52); prepubertal O9 yearsZ81.9 (41.58–155.82); Tanner G2Z35.42 (6.44–156.8); Tanner G3Z9.24 (3.08–102.76); Tanner G4Z6.02 (2.1–15.68) and Tanner G5Z6.86 (3.22–17.92). NA, not available; [, high; Y, low; N, normal. aAndersson AM. Inhibin B in the assessment of seminiferous tubular function. Baillie` re’s Best Practice & Research. Clinical Endocrinology & 2000 14(3) 389–397. bAksglaede L, et al. Changes in anti-Mu¨ llerian hormone (AMH) throughout the life span: a population-based study of 1027 healthy males from birth (cord blood) to the age of 69 years. Journal of Clinical Endocrinology and Metabolism 2010 95(12) 5357–5364. 762 via freeaccess Clinical Study S Tantawy and others SF1 in Egyptians with 46,XY DSD 170:5 763

is predicted to result in conformational change of the sexual activity and fathered two other children. The protein. The arginine residue in position 62 is highly mother showed a normal WT sequence. All 100 Egyptian conserved in all species (Fig. 1). DNA of the parents was controls did not harbour this mutation. not available for genetic analysis.

Single-nucleotide polymorphisms Patient 2 (p.Glu121AlafsX25) " Patient 2 is a male born to non-consanguineous parents. He presented at 13 years Six different SNPs were detected in our patients in both of age with severely hypoplastic phallus (stretched penile heterozygous and homozygous forms: p.Gly146Ala, length was 3.5 cm, !K2.5 SDS) and impalpable right p.Pro125Pro, IVS4-13, c.871-20bp COT, p.Leu451Leu and testis. The left testis was descended and of average size. c.*82 COT. The p.Gly146Ala (c.437GOC, rs1110061) Pelvic laparoscopy revealed atrophic right testis and no polymorphism, was found in 17 patients (34%) and 31 Mu¨llerian structures. He was operated upon for correction controls in this study. We report a frequency of of hypospadias and bifid scrotum. Serum post-hCG p.Gly146Ala in Egypt (31%), which is synonymous with testosterone level was low for age. AMH and inhibin B the global minor allele frequency (27.2%) in the 1000 levels were very low for age and Tanner stage (Table 1). Genome phase 1 population (http://www.1000genomes. Penile length increased to 5.5 cm on testosterone org) from 1094 worldwide individuals, making it a enanthate injections. The patient showed no signs or common polymorphism in Egypt as well (27).Wedetected symptoms of the adrenals being affected. Mutational the heterozygous form of this SNP (Ala/Gly genotype) in analysis revealed a heterozygous p.Glu121AlafsX25 34% of our patients (17/50) and 26% of controls (26/100). (c.361delGAGACAGG) which is an 8 bp deletion mutation Moreover, the homozygous form (Ala/Ala genotype) was causing frameshift resulting in a premature stop codon in found in 5% of controls (5/100) and in none of our patients. exon 4 (Fig. 1), predicted to produce either a severely In previous studies, the SNP p.Gly146Ala was described as truncated protein or no protein at all through nonsense- having slightly reduced transcriptional activity and to be mediated mRNA decay. It is a de novo mutation as none of more common in cryptorchidism (28, 29). the parents harbour it. Furthermore, two novel heterozygous intron 6 mutations (c.1137C105b COT and c.1137C129b COA) Patient 3 (p.Ala154Thr) " Patient 3 is a male patient, born were detected 105 and 129 bp, respectively, following to consanguineous parents, who presented at 4 years of age exon 6. Neither of them was reported in the SNP databases with hypoplastic anchored phallus (stretched penile length (Ensembl, Exome Variant Server). Neither were detected in

European Journal of Endocrinology was 2.5 cm, !K2.5 SDS) and penile hypospadias. Both the control cohort. testes were descended and normal. Hypospadias was surgically corrected. Serum basal testosterone level Functional analysis was normal for age, while post-hCG testosterone level was slightly decreased. LHRH stimulation test revealed slightly Both p.Ala154Thr and p.Arg62Cys show an aberrant elevated FSH level. Inhibin B and AMH levels were normal transactivation of the known SF1-responsive reporters (Table 1). The patient showed no clinical signs or symptoms (human AMH promoter, human CYP11A1 promoter and of the adrenals being affected. Mutational analysis revealed mouse TESCO core enhancer of Sox9) in human embryo- a heterozygous p.Ala154Thr (c.460 GOA) mutation which nic kidney, HEK293-T, cells (Fig. 3). A previously pub- lies in hinge region in exon 4. The hinge region is lished loss of function p.Gly35Glu mutation was used as a important for transcriptional capacity of SF1 as it contains control. The p.Arg62Cys mutant protein shows a signi- a phosphorylation site and a synergy control motif with ficant reduction, whereas p.Ala154Thr shows a significant sumoylation sites and hence is predicted to reduce the increase in its ability to stimulate either of the reporter transcriptional capacity of the protein (24). Both the genes’ activity. The activities of the mutant were nucleotide and the amino acid in this position are calculated relative to that of the WT NR5A1, which was conserved in human, chimp and rhesus but not in other designated as 100%. species (Fig. 1). There were no SNPs found in the altered region as well as no abrogation of potential splice sites. Discussion The heterozygous p.Ala154Thr mutation was inher- ited from the unaffected father. He had normal male A growing number of heterozygous mutations in NR5A1 genitalia (G5, P5, testes 25 ml) and a history of normal have been identified in patients displaying the most

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A 250 CYP11A1 promoter common SF1 phenotype of 46,XY DSD with ambiguous genitalia due to severe partial gonadal dysgenesis at birth 200 (9, 16, 21, 28, 29, 30, 31, 32, 33, 34, 35). Recent studies

150 have reported several cases with NR5A1 mutations and the milder 46,XY DSD phenotype of hypospadias (8, 9, 10, 16) 100 (Fig. 2). We report three novel NR5A1 mutations with % (RLU WT) variable degrees of hypospadias from an Egyptian cohort 50 of patients with 46,XY DSD. An estimated frequency of

0 3–5% has been reported in patients with hypospadias in Europe (8, 10). We found two novel NR5A1 mutations WT Empty with reduced transcriptional function in 23 patients with Arg62Cys p.Gly35Glu p.Ala154Thr hypospadias (8.5%). This rather high frequency might be B 150 TESCO due to preselection of more severe cases (Fig. 2). In 46,XY DSD patients with severe underandrogenisa- tion (female or severely ambiguous external genitalia and 100 cryptorchidism with or without uterus), a frequency of NR5A1 mutations of 8–15% (8, 10, 16) has been reported in Europe. In this study, we investigated 13 patients

% (RLU WT) 50 with a similar phenotype and could not detect any NR5A1 mutations. 0 All three patients with NR5A1 mutations were

WT presenting with penile hypospadias and hypoplastic Empty phallus. Patients 1 and 2 (p.Arg62Cys and p.Glu121A- p.Gly35Glu p.Arg62Cys p.Ala154Thr lafsX25) also displayed maldescended testes and partial C 200 AMH promoter gonadal dysgenesis with impaired and functions (low testosterone, low inhibin B and AMH). 150 Patient 3 (p.Ala154Thr) showed normally descended testes, only mildly impaired Leydig cell and normal Sertoli 100 cell function (slightly decreased testosterone, normal

European Journal of Endocrinology inhibin B and AMH) (Table 1). % (RLU WT) 50 The results of the functional in vitro assays of the mutations of patients 1 and 3 were in accordance with 0 their phenotypes. The p.Arg62Cys mutation of patient 1

WT showed significantly reduced transcriptional activity of Empty the CYP11A1 and AMH promoters and TESCO, while the p.Gly35Glu p.Arg62Cys p.Ala154Thr p.Ala154Thr mutation of patient 3 did not result in reduction of transcriptional activity of any of the three Figure 3 reporters (Fig. 3). Effect of the SF1 mutants (p.Ala154Thr and p.Arg62Cys) on Our patients demonstrated moderate clinical under- transcriptional activities of the CYP11A1 promoter (A), TESCO (B) androgenisation at birth showing that Leydig cell function and the AMH promoter (C). Studies of transcriptional activities of was already disturbed during prenatal life. In contrast, p.Ala154Thr and p.Arg62Cys on the CYP11A1 promoter, TESCO and embryonic Sertoli cell function seemed to have been the AMH promoter were performed in HEK293-T cells. The known sufficient for adequate AMH production and subsequent inactivating p.Gly35Glu mutation was used as control. To compare Mu¨llerian duct regression prenatally. In patients 1 and 2, independent experiments, the activity of the mutants is shown as AMH and inhibin B levels were diminished at 3 and 13 thepercentageoftheSF1WTactivity,whichissetat100%.Data years of age respectively, revealing progressive Sertoli cell represent the mean of two to three independent experiments, each hypofunction as described previously in 46,XY patients performed in 12 replicates. The testosterone bars represent the S.E.M. with NR5A1 mutations and progressive deterioration of Significant outliners (aZ0.05) of the raw data have been eliminated the Sertoli cell function with age (13, 36). Patient 3 using GraphPad Software (graphpad.com/quickcalcs/Grubbs1.cfm). displayed normal Sertoli cell function at 4 years of age.

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To date, two other mutations were reported with isolated We hypothesise that the mild clinical picture of patient 3 hypospadias and normal testes (9, 10). Warman et al. might be attributed to the less critical mutation in the reported a family with six members harbouring a hinge region. However, so far, the p.Ala154Thr mutant did heterozygous NR5A1 hinge region mutation (p.Tyr183X) not show reduced transcriptional activity in vitro. But, and displaying a wide range of different phenotypes. Three interaction of the mutant with modifying factors affecting affected members had isolated hypospadias, two of whom testosterone production through protein interaction were severe perineal and one was mild glandular (9). might play a role in the development of hypospadias in Another case of isolated distal hypospadias carrying a this patient. Further studies are needed to support this de novo heterozygous (p.Arg313Cys) hypothesis. So far, almost only nonsense mutations in the located at the end of the ligand-binding domain of the hinge region have been reported in the DSD phenotypes NR5A1 gene was reported by Allali et al. (10) (Table 2). (9, 10, 16, 17, 21, 30, 34), hypospadias (8, 9), 46,XX So far, no clear genotype–phenotype correlation could primary ovarian insufficiency (16, 17) and premature be detected in patients with NR5A1 mutations. However, ovarian failure (9). In contrast, only missense mutations in patients with severe forms of 46,XY DSD, previously in the hinge region were detected in the minor DSD described mutations are mostly missense mutations in the phenotype of male infertility (13, 14) (Table 2). DNA-binding region (including its accessory DNA-binding Interestingly, the unaffected father of patient 3 domain) or in the ligand-binding domain as well as harboured the same mutation. Previously, another case nonsense mutations leading to severe changes of the was reported with a heterozygous NR5A1 mutation protein (8, 10, 16, 21, 30, 31, 32, 33, 36, 37, 38, 39). (p.Arg281Pro) transmitted by the unaffected father to his son with hypospadias and testicular dysgenesis (40).

Table 2 SF1 mutations described previously in the hinge Mosaicism was suggested to be the reason for the different region and their phenotype. phenotypes in the father and son as the mutation was detected in the father’s DNA at a low copy number SF1 mutations in hinge through direct sequencing and high-resolution melting a Phenotype group region References assay (40). In our case, the father’s and son’s mutations Complete/severe gonadal c.424_427dupCCCA (8) showed similar strength in the sequences (data not dysgenesis female c.666delC (17) shown). However, genetic modifier, thresholds or dosage external genitalia with (or NA) Mu¨ llerian effects of SF1 might contribute to the different phenotypes structures

European Journal of Endocrinology of father and son. Ambiguous genitalia c.536delC (30) The p.Gly146Ala polymorphism, which was described or virilised female p.Y138X (21) external genitalia with c.390delG (10, 17) previously as having reduced transcriptional activity and no (or NA) Mu¨ llerian p.P124PfsX24 (34) being associated with micropenis or cryptorchidism, was structures p.Y183X (9) p.L231_L233dup (16) found to be very frequent in our Egyptian cohort with p.Q206TfsX20 (16) 46,XY DSD and the controls in this study. We detected the p.P130RfsX165 (16) heterozygous form of this SNP (Ala/Gly genotype) in 34% Hypospadias and p.Q107X (8) cryptorchidism of our patients and 26% of controls. Moreover, the SNP Isolated hypospadias p.Y183X (9) was found in a homozygous form (Ala/Ala genotype) in C Male infertility p.G123A p.P129L (13) 5% of controls. The high frequency of p.Gly146Ala in our p.P131L (13) p.R191C (13) controls suggests no pathogenicity compared with the p.G212S (13) previous studies (28, 29). p.D238N (13) This is the first work searching for NR5A1 mutations in p.P210P (14) p.V240V (14) patients from the Middle East and Arab region with high p.G165R (14) consanguinity suggesting a high frequency of NR5A1 46,XX POI c.666delC (17) c.390delG (17) mutations as in European cohorts. p.L231_L233del (17) The type and location of the mutation might be the C p.G123A p.P129L (17) cause of some differences of the phenotypes. However, p.P235L (16) 46,XX POF p.Y183X (9) most probably, other additional modifying factors or digenic mutations should be considered especially in aNone of the patients had adrenal failure. familial cases (35).

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In conclusion, we also recommend the screening and low ovarian reserve in fertile 46,XX subjects. Hormone Research in of NR5A1 in patients with hypospadias and gonadal Paediatrics 2011 75 70–77. (doi:10.1159/000320029) 10 Allali S, Muller JB, Brauner R, Lourenco D, Boudjenah R, Karageorgou V, dysgenesis as well as yearly follow-ups of gonadal function Trivin C, Lottmann H, Lortat-Jacob S, Nihoul-Fekete C et al. Mutation beginning at puberty due to the risk of future fertility analysis of NR5A1 encoding steroidogenic factor 1 in 77 patients with 46, XY disorders of sex development (DSD) including hypospadias. problems. As the clinical consequences are not yet clear, PLoS ONE 2011 6 e24117. (doi:10.1371/journal.pone.0024117) more outcome data are needed before recommending 11 Philibert P, Zenaty D, Lin L, Soskin S, Audran F, Leger J, Achermann JC general screening of NR5A1 in isolated hypospadias. & Sultan C. Mutational analysis of steroidogenic factor 1 (NR5a1) in 24 boys with bilateral anorchia: a French collaborative study. Human However, future mild progressive effects on the 2007 22 3255–3261. (doi:10.1093/humrep/dem278) gonadals resulting in male infertility cannot be excluded 12 Paris F, De Ferran K, Bhangoo A, Ten S, Lahlou N, Audran F, Servant N, in this case, as also infertile males with normal male Poulat F, Philibert P & Sultan C. Isolated ‘idiopathic’ micropenis: hidden genetic defects? International Journal of Andrology 2011 34 phenotypes and NR5A1 mutations were described recently e518–e525. (doi:10.1111/j.1365-2605.2010.01135.x) (13, 14). 13 Bashamboo A & McElreavey K. NR5A1/SF-1 and development and function of the . Annales d’Endocrinologie 2010 71 177–182. (doi:10.1016/j.ando.2010.02.013) 14 Ropke A, Tewes AC, Gromoll J, Kliesch S, Wieacker P & Tuttelmann F. 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Received 27 November 2013 European Journal of Endocrinology Revised version received 18 February 2014 Accepted 3 March 2014

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